Detection and identification of simple nucleotide mutations and/or polymorphisms among individuals are very important in many biological fields, ranging from biomedical research in hereditary diseases to ecology and evolutionary biology. Identification of the location of mutations involved in heritable diseases can provide clues for the diagnosis, prognosis and therapeutic treatment of such diseases. However, it is very difficult to localize most of the mutations into very small region (several kilo-base pairs) or a single gene at a genomic level.
Sequencing of the genome of different individuals is the most straightforward method for mutation detection, but it is expensive and time-consuming. Some methods have been developed to detect mutations without direct sequencing. Linkage and association mapping use polymorphic markers to approximate the chromosomal location of the mutations. This method is very efficient to localize a mutation into a quite large genomic region (several hundreds kilo-base pairs), but very slow to further localize it into a single gene or a small region (several kilo-base pairs). Restriction fragment length polymorphism (RFLP) is good for mutation detection and sequence comparison but can not efficiently define the location of a mutation either if whole genomic DNA is used as the starting material.
Most of the currently developed methods or techniques are suitable for testing mutations in a small region or a handful of genes, but are not good (if not impossible) for localizing mutations or polymorphisms at a genomic level. For example, in polynucleotide microarray hybridization methods (e.g. U.S. Pat. No. 5,837,832 (Chee et al.), and U.S. Pat. No. 6,376,191 (Yu et al.)) mutations in every single base position are detected by a set of four primers. To fully detect a 10 kb region, these methods will need about 10,000 sets of primers to be spotted on a microarray slide. Single stranded conformational polymorphism (SSCP) and denaturing gradient gel electrophoresis (DGGE) (Orita et al (1989) PNAS 86:2766; Myers et al (1985) N. A. R. 13:3131; White et al (1992) Genomics 5:301; Mills et al (1994) Biochem. 33:1797) methods are based on the observations that DNA sequence variations can cause DNA electrophoretic mobility changes. These two methods only work with short DNA molecules and can not screen many genes simultaneously.
The two methods I present in this invention have great potential to detect and localize DNA mutations at a genomic level simultaneously and rapidly. The method using restriction endonuclease(s) to detect mutations is termed RE microarray method. The other method using mismatch-recognition endonuclease(s) is named MR microarray method. The principle of the methods is illustrated in FIG. 1.